DESCRIPTION (adapted from the application) Lack of permanence is a major problem with current gene therapy vectors. While retrovirus vectors attain permanence by integrating into chromosomes, these vectors integrate randomly, leading to mutagenesis and loss of predictability of gene expression. Most other vectors fail to integrate efficiently and are lost over time. We wish to develop a method to provide permanent gene therapy by chromosomal integration of the gene of interest in a site-specific and efficient manner. To supply these attributes, we are using a recombinase enzyme that can achieve precise recombination at a high efficiency, restricted to its own recognition site. In particular, we are using the CRE recombines. Integration will occur only at pre-chosen sites in mammalian genomes that have sufficient homology to the native LOX sites to be used efficiently in recombination by the CRE enzyme. We will find such pseudo LOX sites in the genome database by computer, then develop rapid assays to evaluate their recombination frequencies. Those sites mediating efficient recombination in rapid assays in bacteria and human cells will be analyzed for site-specific integration into the chromosomes. A CRE expression system optimized for integration, and minimizing excision, will be developed. The pilot system will achieve efficient, permanent, site-specific integration at defined, well- expressed sites in the chromosomes of human and mouse cells. This system can be used in combination with viral or non-viral gene delivery methods. The next experiments will use such methods to test integration in mice, preparatory to testing in patients.